Arresting cell growth with novel functionalised indolocarbazoles - PowerPoint PPT Presentation

Arresting cell growth with novel functionalised indolocarbazoles Florence O. McCarthy 1, *, Hannah J. Winfield 1 , Kevin D. O’Shea 1 , Michael M. Cahill 1 and Larry T. Pierce 1 1 Department of Chemistry and ABCRF, Cavanagh Building, University College Cork, Western Road, Cork, Ireland • Corresponding author: f.mccarthy@ucc.ie 1

Arresting cell growth with novel functionalised indolocarbazoles Graphical Abstract M M – Messenger I – Inhibitor I M Kinase Modification Identification 2

Abstract: Cancer causes about 13% of all human deaths and at least one fifth of all deaths in Europe and North America. Although chemotherapy is increasingly prescribed, it is not without side effects and so new, more selective remedies for cancer sufferers must be found. Since the discovery of the anticancer properties of the indolocarbazole staurosporine, many analogues have been synthesised in order to obtain compounds that have a higher potency with respect to anticancer mechanisms. The overall objective of this project is to produce selective and highly potent novel anticancer agents through modification of the indolocarbazole structure and a focus of this work is the replacement of the lactam/maleimide heretocycle to form a series of novel indolocarbazole derivatives including the first reported synthesis of a series of novel substituted indolocarbazole uracils. Biological evaluation via the NCI 60 cell line screen has been completed for a number of these compounds with some showing significant selectivity towards individual leukaemia and melanoma cell lines. Keywords: indolocarbazole; cancer; kinase; topoisomerase 3

Cancer and Chemotherapy  Over 3.2 million people in Europe diagnosed with cancer on annual basis.  Cumulative lifetime risk of invasive cancer in Ireland is approximately 1 in 3 for men and 1 in 4 for women.  Greater need than ever to pursue targeted cancer therapies via novel drug templates.  Indolo[2,3- a ]carbazole (ICZ) pharmacophore has been a major focus to medicinal chemists for over 30 years.  Staurosporine (STA) first ICZ to be isolated from a natural source; reported by Omura et al . in 1977. 1  Subsequently shown to be an extraordinarily potent inhibitor of PKC (IC 50 = 2.7 nM) and strongly cytotoxic against cancer cells. 2 Omura, S. et al., J. Antibiot., 1977 , 30, 275 1 Tamaoki, T. et al., Biochem. Biophys. Res. Commun. , 1986 , 135, 397 2

Indolo[2,3- a ]carbazoles as protein kinase inhibitors  One of the largest families of proteins in humans, deregulation of protein kinases has been implicated in oncogenesis and the progression of tumours.  Oncogenic kinases continuously activate signalling pathways that regulate cell cycle progression, proliferation and cell survival.  STA found to be a nonselective inhibitor of many different kinases, such as PKA (IC 50 = 15 nM), phosphorylase kinase (IC 50 = 3 nM) and S6 kinase (IC 50 = 5 nM). 3  Crystal structures resolved for STA in complex with cyclin-dependent kinase 2 (CDK2) and PKA proved inhibition occurs in an ATP-competitive manner. 4,5  Although ATP-binding pocket is relatively conserved across pan-kinase domain, exploitation of discreet differences in active site residues and conformations can help to confer selectivity. Meggio, F. et al., Eur. J. Biochem, 1995 , 234, 317 3 STA in complex with CDK2 Lydon, N. et al., Structure , 1997 , 5, 1551 4 Engh, R.A. et al., Structure , 1997 , 5, 1627 5

Staurosporine: as a lead for kinase inhibition

Rebeccamycin: another lead ICZ candidate Topo I cleavage complex with human DNA  Rebeccamycin (REB), an ICZ with one N -glycosidic bond, was isolated in 1985 from Nocardia aerocolonigenes . 6  REB displayed considerable activity against leukemia and melanoma in mice, and inhibited the growth of A549 human lung adenocarcinoma cells, producing single strand breaks in the DNA of these cells. 7  Potent anticancer action was linked to its inhibition of topoisomerase I (topo I). Clardy, J. et al., Tet. Lett., 1985 , 26, 4011 6 Tomita, K. et al., J. Antibiot. , 1987 , 40, 668 7

Bisindolylmaleimides: potent ICZ precursors  Bisindolylmaleimides (BIMs) are frequently utilised as synthetic precursors to ICZs, with numerous coupling methods employed to achieve final aromatisation step.  Also found to possess uniquely potent biological activity, and a number of candidates are under consideration for the treatment of diseases such as non-small cell lung cancer, glioblastoma and diabetic peripheral retinopathy. Kuo, G.-H. et al., J. Med. Chem. , 2003 , 46, 4021 8

Further diversification of the ICZ pharmacophore  Appropriation of heteroaryl subunits in place of one indole functionality has been shown to increase kinase inhibition in many instances. Meggers, E. et al., Synthesis, 2005 , 9, 1521 9 Peifer, C. et al., J. Med. Chem. , 2006 , 49, 7549 10

Target Indolocarbazoles  Literature suggests that there is significant scope to modify the indolocarbazole template and maintain biological activity but imbue differentiation of mode of action.  One area that has been relatively overlooked has been the F-ring and this is the focus of our current work. M M – Messenger I – Inhibitor I M Kinase Modification Staurosporine  A common F-ring motif in reported biologically active indolocarbazoles is the lactam/maleimide. We seek to alter the H-bonding framework to isolate new targets.  Our work to date has focussed on utilising several novel 5- and 6-membered heterocycles (X-Y-Z) to replace this ring with Target Structure unique biological profiles.

Aims and objectives • The primary aim of our program of diversity-oriented synthesis is to explore the paradigm of F-ring modulation in novel indolocarbazoles and azaindolocarbazoles. • It is envisaged that such modification can help to confer more favourable pharmacological properties and potentially increase bioavailability • Evaluation undertaken by assessment of cell growth and consequently the influence of these novel templates in the topo I-DNA complex and the exploitation of discrete differences in the kinase active site. • Initial evaluation of antiproliferative activity is followed by further investigation of discrete biological mechanism of action.

Diversity Orientated Synthesis  Designed synthesis via a versatile key intermediate Bisindolyl b -Keto ester   Subsequent modification to give a series of novel bisindole heterocycles  Adaptable route provides access to 5- and 6-membered rings  Cyclisation to final indolocarbazoles reported for the first time  Starting from indole or 7-azaindole will give rise to indolocarbazoles and azaindolocarbazoles

Initial synthesis of b -keto ester intermediate Formation of the fully protected b -  keto ester proceeds smoothly  However, all attempts at pyrimidinedione formation with urea condensation fail, despite multiple conditions including microwave.

Pyrimidine-2,4-dione synthesis

Pyrimidine-2,4-dione synthesis  Synthesis of more robust methyl protected b -keto ester proceeds smoothly  Initial attempts at pyrimidinedione formation again fail, but successful on changing to thiourea.  Can be converted to uracil 11. L.T. Pierce, M.M. Cahill & F. O. McCarthy Tetrahedron 2010 , 66(51), 9754-9761

Exploring novel bisindolyl heterocycles  The thiouracil is a good template for further derivatisation  Thiophilic substitution and novel ring formation are both possible.  Removal of the sulfur can also be effected in a facile manner. 11. L.T. Pierce, M.M. Cahill & F. O. McCarthy Tetrahedron 2010 , 66(51), 9754-9761

Bisindolyl pyrazolones/aminopyrimidinones Modulating H-bonding character 47 48 42  Use of hydrazine as nucleophile yields pyrazolones which can again be functionalised further  49 Guanidine in place of thiourea is also successful in 6-membered ring formation. 50

Bisindolyl pyrimidinone cyclisation study Table 1 Conditions investigated for the oxidative cyclisation of bisindolyl precursor to novel aromatized indolocarbazole a Reagent Amount Conditions Reaction time Product DMF, 130  C b Pd(OAc) 2 1.0 equiv 20h - AcOH, 110  C c Pd(OAc) 2 5.0 equiv 24h - DMF, 100  C b Pd(CF 3 CO 2 ) 2 3.0 equiv 20h - H 2 O/ KOH, 100  C c K 3 [Fe(CN) 6 ] 1.0 equiv 24h - DCM, r.t c PhI(OAc) 2 2.5 equiv 36h - hυ / I 2 1.0 equiv d toluene, r.t c 72h SM/Product hυ / I 2 CH 3 CN/MeOH (3:2) b catalytic 24h - hυ / I 2 CH 3 CN/MeOH (3:2) c,f catalytic 16h Product (53%) hυ / I 2 catalytic CH 3 CN/MeOH (3:2) e,f 16h Product (55%) a Reactions were performed on 0.27 mmol scale. b Inert atmosphere. c Open-vessel reaction. d Refers to stoichiometry of iodine. e Air-bubbling f Dilution: 1.0 mg substrate/ 2.5 mL solvent.  Indolocarbazole formation from bisindolemaleimide precursors is well described in the literature.  Specific conditions are required once the maleimide has been converted to another heterocycle. 12. L.T. Pierce, M.M. Cahill, H.J. Winfield & F. O. McCarthy Eur.J.Med.Chem 2012 , 56, 292-300

Azaindole b -Keto ester and pyrazolone formation  Temperature control and solubility critical to success Azaindole b -keto ester  formation is temperature dependant due to solubilty.  Use of hydrazine forms the pyrazolone in good yield.